DsPETase01 plastic degrading enzyme mutant and application thereof

Abstract

The invention discloses a dsPETase01 plastic degrading enzyme mutant and application thereof, and relates to the technical field of bioengineering, the amino acid sequence of a wild type plastic degrading enzyme (dsPETase01) is designed and modified, 8 dsPETase01 single mutation sites or 19 combined mutation sites are provided, the thermal stability of the obtained dsPETase01 mutant is improved by about 4-21 DEG C compared with that of the wild type dsPETase01, and the dsPETase01 mutant can be used for preparing a plastic degrading enzyme. And the activity is improved by nearly 1.2-3 times. The invention also provides four preferable mutant proteins which have higher yield, activity and thermal stability compared with wild dsPETase01, have wider application conditions, are more suitable for degrading PET (Polyethylene Terephthalate) plastics, and are beneficial to large-scale production and industrial application.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a dsPETase01 plastic-degrading enzyme mutant and its application. Background Art

[0002] Ethylene terephthalate (PET) is the most important variety of thermoplastic polyester, commonly known as polyester resin, which is white or light yellow with a smooth and shiny surface. It has excellent physical and mechanical properties within a relatively wide temperature range, and the service temperature can reach 120 °C. It has excellent electrical insulation properties, and even at high temperatures and high frequencies, its electrical properties are still good. Therefore, it is widely used in the fields of packaging, electronics and electrical appliances, medical and health, construction, and automobiles, accounting for 1 / 6 of the total amount of all plastics. With the increasing use of PET, the subsequent problem is the pollution problem of PET plastics. These plastics are difficult to degrade in the natural environment, resulting in the accumulation of a large amount of plastic waste, which has brought long-term and profound impacts on the ecological environment and human health.

[0003] More and more researchers have found that certain microorganisms and enzymes have potential in the process of plastic degradation. For the bioenzymes that can degrade PET plastics, they were discovered relatively late. The most classic plastic-degrading enzyme LCC that can degrade PET was discovered in leaf and twig compost cutin. This enzyme has achieved some industrial applications. Some other PET plastic-degrading enzymes such as isPETase that eats plastics in 2016 and PHL7 isolated in 2022. These enzymes all have the potential to decompose PET, but due to their low thermal stability, it is difficult to achieve industrial degradation of PET plastics. Summary of the Invention

[0004] The purpose of the present invention is to provide a dsPETase01 plastic-degrading enzyme mutant and its application.

[0005] The present invention realizes the above purpose through the following technical solutions:

[0006] As the first aspect of the present invention, a dsPETase01 plastic-degrading enzyme mutant is provided. The dsPETase01 plastic-degrading enzyme mutant is obtained by performing single-point mutation, combined mutation, or introducing a disulfide bond into the single-point mutation sequence or combined mutation sequence of the wild-type dsPETase01 plastic-degrading enzyme sequence shown in SEQ ID NO.1;

[0007] Among them, the single mutation site includes at least one of Q171L, G191E, G191D, G191R, G191Q, S215P, A242V, or F249A;

[0008] The combination of mutation sites includes at least one group of (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, A242V), (K142R, S215P), (K142R, A242V), (E2P, A242V), (S215P, A242V), (E2P, K142R, Q171L, G191E), (K142R, Q171L, G191E), (E2P, K142R, Q171L, G191E, A242V), (E2P, K142R, Q171L, G191E, S215P), (E2P, K142R, Q171L, G191E, S215P, A242V), (Q171L, G191E, S215P, Q171L, S215P, K142R) or (Q171L, G191E, S215P, A242V).

[0009] As a further optimization scheme of the present invention, the mutation sites of the dsPETase01 plastic-degrading enzyme mutant are (Q171L, G191E) or (Q171L, G191E, S215P) or (Q171L, G191E, S215P, A242V). The amino acid sequences of the dsPETase01 plastic-degrading enzyme mutants obtained by the above mutations are shown in SEQ ID NO.2-4.

[0010] As a further optimization scheme of the present invention, the amino acid sequence of the dsPETase01 plastic-degrading enzyme mutant obtained by introducing disulfide bond modification on the sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.5.

[0011] As a second aspect of the present invention, a polynucleotide is also provided, and the polynucleotide encodes the dsPETase01 plastic-degrading enzyme mutant as described in any one of the above.

[0012] As a further optimization scheme of the present invention, the polynucleotide sequences encoding the amino acid sequences of the dsPETase01 plastic-degrading enzyme mutants shown in SEQ ID NO.2-5 are shown in SEQ ID NO.6-9 respectively.

[0013] As a third aspect of the present invention, a recombinant plasmid is also provided. The recombinant plasmid is an expression vector capable of corresponding translation and expression of the dsPETase01 plastic-degrading enzyme mutant as described in any one of the above, and the expression vector is a pET-28a vector.

[0014] As the fourth aspect of the present invention, there is also provided an application of the dsPETase01 plastic-degrading enzyme mutant as described in any one of the above in degrading or recycling PET plastics.

[0015] As a further optimized solution of the present invention, the application is that the dsPETase01 plastic-degrading enzyme mutant decomposes PET plastics and generates terephthalic acid TPA, the main component of PET plastics, during the decomposition process.

[0016] The present invention has the following beneficial effects:

[0017] The present invention takes the wild-type dsPETase01 plastic-degrading enzyme derived from the deep sea (Chen, J., Jia, Y., Sun, Y. et al. Global marine microbial diversity and its potential in bioprospecting. Nature 633, 371–379 (2024).) as the research object, designs and modifies its amino acid sequence, and provides a series of single-point mutations and combined mutations for the wild-type dsPETase01 plastic-degrading enzyme. The thermal stability of these mutant proteins is increased by about 4 - 21 °C compared with the wild-type dsPETase01 plastic-degrading enzyme, and the activity is increased by nearly 1.2 - 3 times. The dsPETase01 plastic-degrading enzyme mutant has higher yield, activity and thermal stability compared with the wild-type dsPETase01 plastic-degrading enzyme, is more suitable for industrial plastic degradation, and is conducive to large-scale production and industrial application. Description of the Drawings

[0018] Figure 1 It is the map of the modified pET-28a vector;

[0019] Figure 2A 、 Figure 2B 、 Figure 2C It is the small-scale purification result of the dsPETase01 single mutant protein;

[0020] Figure 3A 、 Figure 3B It is the small-scale purification result of the dsPETase01 double mutant protein;

[0021] Figure 4 It is the small-scale purification result of the dsPETase01 combined mutant protein;

[0022] Figure 5 It is the chemical structural formula of PET and BHET-OH;

[0023] Figure 6A 、 Figure 6BResults of activity assays of dsPETase01 single mutant proteins, dsPETase01 double mutant proteins, and dsPETase01 combined mutant proteins;

[0024] Figure 7 Results of affinity chromatography purification of dsPETase01 high-quality mutant proteins;

[0025] Figure 8A 、 Figure 8B 、 Figure 8C Results of quality assays of dsPETase01 high-quality mutant proteins.

[0026] Figure 9 Results of activity identification of dsPETase01 high-quality mutant proteins. Detailed implementation manners

[0027] The present application will be further described in detail below with reference to the accompanying drawings. It is necessary to point out here that the following detailed implementation manners are only used to further illustrate the present application and should not be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0028] 1. Materials and reagents

[0029] The methods used in the present invention are all conventional methods known to those skilled in the art unless otherwise specified. For those without specific conditions, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] 2. Methods

[0031] 2.1 Construction of dsPETase01 mutant plasmids

[0032] The gene sequences of wild-type dsPETase01 and its mutants were obtained by gene synthesis. The protein sequence of wild-type dsPETase01 is shown in SEQ ID NO.1. All mutants were constructed based on the wild-type by designing corresponding mutant primers according to the method of molecular cloning, including 27 single-point mutations: D4P, Q171L, Q109Y, G191E, G191D, H44Y, G191R, G191Q, T143A, K142R, E2P, F33S, S13A, E148R, E6L, E6V, S215P, Q259E, T154A, E67Q, A242V, S26A, F249A, K160R, S254A, E139Y, G191A.

[0033] A total of 15 mutant proteins with double mutations (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, S215P), (G191E, A242V), (E2P, K142R), (K142R, S215P), (K142R, A242V), (E2P, S215P), (E2P, A242V), (S215P, A242V).

[0034] Combined mutations: (E2P, K142R, Q171L, G191E), (K142R, Q171L, G191E), (E2P, K142R, Q171L, G191E, A242V), (E2P, K142R, Q171L, G191E, S215P), (E2P, K142R, Q171L, G191E, S215P, A242V), (Q171L, G191E, S215P), (Q171L, S215P, K142R), (Q171L, G191E, S215P, A242V), a total of 8 combined mutations.

[0035] Wild-type dsPETase01 and its mutant proteins were all constructed on the modified pET-28a vector (GenScript). An 8His-strepII-TEV-GG tag sequence was fused behind the T7 promoter of this vector. The tag sequence is shown in SEQ ID NO.10 (where 8His and strep II are tag sequences for affinity purification, "TEV" is the TEV protease cleavage site for removing the tag during subsequent purification, and "GG" is the tag sequence). The gene sequences of the constructed recombinant proteins were all verified to be correct by a sequencing company. The vector map is shown in Figure 1 。

[0036] 2.2 Small-scale expression and purification of dsPETase01 mutant proteins

[0037] Using conventional molecular biology methods, the constructed dsPETase01 mutant plasmids were respectively transformed into BL21(DE3) Escherichia coli competent cells in a laminar flow cabinet and cultured overnight at 37°C. Single colonies from the overnight culture were picked into 5 ml of LB liquid medium and cultured at 37°C until the OD of the bacterial solution 600When it is 0.6 - 0.8, take a small amount of bacterial liquid and fix it with loading buffer. Take a small amount of bacterial liquid, add glycerol and freeze it at -80 °C. Add 0.5 mM IPTG to the remaining bacterial liquid, incubate at 15 °C for 16 hours, collect the bacterial cells, and take the induced bacterial liquid for SDS-PAGE detection. Dissolve the collected bacterial cells with lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol), then break them by sonication, centrifuge at 12000 rpm at 4 °C for 10 minutes, collect the supernatant, and add the supernatant to 50 μL of Strep- XT packing material that has been treated with buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol), incubate at 4 °C for 30 minutes. For the incubated sample, centrifuge at 12000 rpm at 4 °C for 10 minutes, add 1 mL of buffer, wash 3 times, then add 100 μL of elution buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 75 mM biotin), centrifuge at 12000 rpm, 4 °C for 5 minutes, and collect the eluted sample. Take a small amount of the sample from each step of the treatment, fix it with loading buffer, and perform SDS-PAGE detection.

[0038] The experimental results are shown in Figure 2A 、 Figure 2B and Figure 2C . Among the 27 single-point mutations, all single-point mutant proteins were significantly expressed. After purifying all the expressed samples, it was found that all mutant proteins could be eluted and had high purity.

[0039] The small-scale test results of double mutations and combined mutants were similar to those of single mutations. All double mutant and combined mutant proteins could be eluted and had high purity. The small-scale results of double mutations are shown in Figure 3A and 3B ; The elution results of combined mutations are shown in Figure 4 .

[0040] 2.3 Detection of the thermal stability of dsPETase01 mutant proteins

[0041] For the detection of the thermal stability of dsPETase01 mutant proteins, the detection technique of micro differential scanning fluorimetry (nano Differential Scanning Fluorimetry, nanoDSF) was selected. The specific operation is as follows:

[0042] Take 20 μL of the protein with a concentration of 0.5 mg / ml and add it to a 384-well experimental plate. After shaking and centrifuging (to avoid uneven samples or aspirating air bubbles during the sampling process), place the experimental plate on the sampling rack, and use a Nano DSF capillary to aspirate the sample to ensure that the sample fills the entire capillary. Place the capillary into the nanoDSF instrument, set the initial temperature to 20 °C, and finally raise the temperature to 90 °C at a rate of 2.0 °C per minute to terminate. The instrument will heat up and perform real-time detection according to the set parameters. The specific numerical results of the Tm values measured for all single mutant proteins are shown in Table 1.

[0043] Table 1. Tm values of dsPETase01 single mutant proteins

[0044]

[0045] As shown in Table 1, among the 27 single mutant proteins, the Tm values of Q171L, G191E, G191D, G191R, G191Q, S215P, A242V, and F249A increased by 2.02 °C - 9.27 °C, and the mutations at the G191 position in 4 of them all led to a significant increase in the Tm value. Therefore, the present invention screened out 8 mutants, namely Q171L, G191E, G191D, G191R, G191Q, S215P, A242V, and F249A, which can improve the thermal stability of dsPETase01.

[0046] The single mutants with significantly increased Tm values were combined and obtained 15 double mutant proteins according to the small-scale expression scheme of single mutants. The Tm values of the specifically detected double mutant proteins are shown in Table 2.

[0047] Table 2. Tm values of dsPETase01 double mutant proteins

[0048]

[0049] As can be seen from Table 2, except for the three double mutants (G191E, S215P), (E2P, K142R), and (E2P, S215P) whose Tm values decreased, the Tm values of the other double mutants all increased significantly, with the increase ranging from 3.85°C to 16.03°C. Therefore, a total of 12 double mutant proteins with improved thermal stability were obtained, namely (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, A242V), (K142R, S215P), (K142R, A242V), (E2P, A242V), and (S215P, A242V). Among them, the double mutant (Q171L, G191E) had the best effect, with its Tm increasing by 16.03°C.

[0050] To further obtain mutants with higher stability, single mutants or double mutants with significantly increased Tm values were combined, and a total of 8 combined mutants were constructed. The Tm values of the specifically detected combined mutant proteins are shown in Table 3.

[0051] Table 3. Tm values of dsPETase01 combined mutant proteins

[0052]

[0053] As can be seen from Table 3, the Tm values of all combined mutants are 10.27°C - 21.17°C higher than that of the wild type. Among them, the Tm values of the mutants (E2P, K142R, Q171L, G191E, S215P, A242V) and (Q171L, G191E, S215P, A242V) both increased by more than 21°C.

[0054] 2.4. Detection of the activity of dsPETase01 mutant proteins

[0055] PET plastic is actually a polyethylene terephthalate, and its structural formula is shown in Figure 5 the left. In the present invention, its smallest unit, bis(2-hydroxyethyl) 2-hydroxyterephthalate (BHET-OH), whose structural formula is shown in Figure 5 the right (Biortus), was used as the substrate to establish an activity measurement protocol for dsPETase01 enzyme. The specific operation is as follows:

[0056] Prepare buffer: 75 mM PBS, pH 8.0, substrate is 2.5 mM BHET-OH (dissolved in DMSO and then added with 10 mM sodium carbonate solution), reaction temperature is 30 °C. Dilute the dsPETase01 mutant protein obtained in step 2.2 to 2 μM with buffer respectively. Transfer 20 μL of the substrate to a 384-well plate and set two replicates, transfer 40 μL of the dsPETase01 mutant protein to be tested to the corresponding well plate, centrifuge immediately and mix well by oscillation, and collect the fluorescence signal values generated by the reaction under the condition of a TECAN F200 microplate reader. Use GraphPad Prism9 analysis software for data analysis, and finally obtain the enzyme activity parameters of the protease to be tested.

[0057] Among them, the activity results of the single mutants are as Figure 6A shown. Among the 27 single mutants detected, the activities of the two mutants E148R and E148K were lost by about 50%, and the activities of other mutants were not much different from those of the wild-type protein, basically between 80% and 127%.

[0058] The activity data of double mutants and combined mutants are shown in Figure 6B , the activity of the double mutants basically decreased by 10%-15% compared with that of the wild-type, and still maintained a relatively high activity. The activity of the combined mutants was basically equivalent to that of the wild-type, indicating that these mutants with improved thermal stability still maintained good activity.

[0059] 2.5 Expression and purification of high-quality mutant proteins of dsPETase01

[0060] In order to further study the function of the high-quality mutant proteins of dsPETase01, the double mutant proteins (Q171L, G191E) with obvious increase in Tm value and activity were selected. Their amino acid sequences are shown in SEQ ID NO.2 respectively, and the nucleotide sequences are shown in SEQ ID NO.6. The combined mutant proteins (Q171L, G191E, S215P), (Q171L, G191E, S215P, A242V) have amino acid sequences shown in SEQ ID NO.3-SEQ ID NO.4 and nucleotide sequences shown in SEQ ID NO.7-SEQ ID NO.8.

[0061] Introducing disulfide bonds to improve the stability of proteins is a common method. The present invention also provides a protein dsPETase01-dis M1 obtained by introducing disulfide bond modification to wild-type dsPETase01 and introducing the double mutant (Q171L, G191E). Its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.9.

[0062] The protein properties of the above proteins were compared. All plasmids were heterologously expressed in E. coli, and the expressed proteins were purified.

[0063] 2.5.1. Purification of dsPETase01 high-quality mutant protein

[0064] (1) Affinity chromatography

[0065] The collected bacterial pellets were weighed respectively, and the corresponding volume of lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) was added according to a ratio of 1:10. The bacterial cells were disrupted using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16,000 rpm. All recombinant dsPETase01 high-quality mutant proteins have a StrepII tag, and the Strep-Tactin XT affinity chromatography column was used to enrich and purify the proteins. The specific procedure was as follows: First, the Strep-Tactin XT affinity chromatography column was washed and equilibrated with the lysis buffer for 10 column volumes. Then, the lysate supernatant was loaded onto the Strep-Tactin XT FF affinity chromatography column, and elution was performed with the lysis buffer containing 75 mM biotin. The eluted proteins were collected for SDS-PAGE detection, and the protein concentration was measured using Nanodrop to calculate the protein yield.

[0066] The results of protein purification are as Figure 7 , the yield of the wild type was 6 mg / L, and the yields of (Q171L, G191E), (Q171L, G191E, S215P), and (Q171L, G191E, S215P, A242V) were 13 mg / L, 15.2 mg / L, and 15.6 mg / L respectively; the yield of dsPETase01-disM1 was 10 mg / L. The yields of all mutants were approximately 2 - 2.6 times higher than that of the wild type.

[0067] (2) Enzyme digestion and reverse affinity chromatography

[0068] To obtain proteins with higher purity, a certain amount of TEV enzyme was added to the sample after the above affinity chromatography. After overnight digestion at 4°C, the digested supernatant was further purified using a HisFF chromatography column. Since the wild type of dsPETase01 and its mutants have no affinity tags after enzyme digestion, they will not bind to the affinity column and will flow out of the column (recorded as the breakthrough fraction), so the breakthrough fraction was collected.

[0069] (3) Gel filtration chromatography and QC detection

[0070] After concentrating the above penetration fluid to about 2 mL, gel filtration chromatography was carried out separately. The model of the gel chromatography column was: Superdex 200 Increase 10 / 300GL, and the buffer for gel chromatography was: 20 mM Tris–HCl pH7.5, 150 mM NaCl, 1 mM DTT. The samples after gel filtration chromatography were collected and the protein content was detected respectively, that is, SDS-PAGE purity detection, mass spectrometry analysis and analytical molecular sieve detection were carried out respectively;

[0071] According to the SDS-PAGE results, the purity of both the wild-type and its dsPETase01 mutant proteins was greater than 99%. The mass spectrometry detection results also showed that the molecular weight of the detected samples was basically consistent with that of the target protein, indicating that the purified protein was the target protein. In addition, the results of the analytical molecular sieve showed that all proteins were in the monomer state in solution. The detection results are shown in Figure 8.

[0072] 2.5.3 Detection of the activity of the dsPETase01 high-quality mutant protein

[0073] In order to further verify the ability of the purified dsPETase01 high-quality mutant protein to degrade plastics, the purified protein was subjected to activity detection again, and the detection method and steps were the same as those in 2.4. The difference was that the protein used for detection was a protein without a tag and had a higher purity. The activity results were as follows: Its activity was detected at 30 °C and 60 °C respectively. The detection results showed that: at 30 °C, the activity of most dsPETase01 high-quality mutants was comparable to that of the wild-type, and among them (Q171L, G191E, S215P) was about 2.1 times higher than the wild-type; at 60 °C, the activity of the mutants was higher than that of the wild-type, and most of them were about 1.5 times higher than the wild-type, and among them (Q171L, G191E, S215P) had an activity about 3 times higher than the wild-type ( Figure 9 ). High-temperature conditions are required for plastic degradation in industry. These mutants not only improve their thermal stability but also improve their activity, indicating that they are more suitable for industrial plastic degradation.

[0074] 3. Conclusion

[0075] The above shows that the dsPETase01 mutant protein provided by the present invention has higher protein yield, higher enzyme activity and better thermal stability, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0076] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A dsPETase01 plastic degrading enzyme mutant, characterized in that: The dsPETase01 plastic degrading enzyme mutant is obtained by subjecting the sequence of the wild-type dsPETase01 plastic degrading enzyme shown in SEQ ID NO.1 to single-point mutation, combined mutation, or introduction of a disulfide bond into the sequence of the single-point mutation or combined mutation; Wherein, the single mutation site includes at least one of Q171L, G191E, G191D, G191R, G191Q, S215P, A242V or F249A; The combined mutation sites at least include (Q171L, G191E), (K142R, Q171L), (E2P, Q171L), (Q171L, S215P), (Q171L, A242V), (K142R, G191E), (E2P, G191E), (G191E, A242V), (K142R, S215P), (K142R, A242V), (E2P, A242V), (S215P, A242V), (E2P, K142R, Q171L, G191E), (K142R, Q171L, G191E), (E2P, K142R, Q171L, G191E, A242V), (E2P, K142R, Q171L, G191E, S215P), (E2P, K142R, Q171L, G191E, S215P, A242V), (Q171L, G191E, S215P, Q171L, S215P, K142R) or (Q171L, G191E, S215P, A242V).

2. A dsPETase01 plastic degrading enzyme mutant according to claim 1, characterized in that: The mutation site of the dsPETase01 plastic degrading enzyme mutant is (Q171L, G191E) or (Q171L, G191E, S215P) or (Q171L, G191E, S215P, A242V). The amino acid sequence of the dsPETase01 plastic degrading enzyme mutant obtained by the above mutation is shown in SEQ ID NO.2-4.

3. A dsPETase01 plastic degrading enzyme mutant according to claim 2, characterized in that: The amino acid sequence of the dsPETase01 plastic degrading enzyme mutant obtained by introducing a disulfide bond into the sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.

5.

4. A polynucleotide, characterized in that The polynucleotide encodes the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1-3.

5. A polynucleotide according to claim 4, characterized in that The polynucleotide sequences encoding the amino acid sequences of the dsPETase01 plastic degrading enzyme mutants shown in SEQ ID NOs. 2-5 are shown in SEQ ID NOs. 6-9, respectively.

6. A recombinant plasmid, characterized in that: The recombinant plasmid is an expression vector capable of correspondingly translating and expressing the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1 to 3, and the expression vector is a pET-28a vector.

7. Use of the dsPETase01 plastic degrading enzyme mutant as described in any one of claims 1 to 3 in the degradation or recycling of PET plastics.

8. The use according to claim 8, characterized in that: The application is to decompose PET plastic by using the dsPETase01 plastic degradation enzyme mutant and generate terephthalic acid TPA, the main component of PET plastic, during the decomposition process.

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